The GI mucosa contains a complex mixture of immune and non-immune cells and their products that maintain a dynamic balance to protect the host against enteric pathogens and at the same time maintain tolerance towards harmless products of digestion and the normal bacterial flora. There is now substantial evidence that the human inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are multifactorial. Two of these factors are proper balance of the mucosal immune system and the microbial flora of the GI tract. The central hypothesis of this proposal is that specific products of enteric microbial flora contribute to the pathogenesis of IBD by inhibiting lymphocyte function thus causing imbalance of the mucosal immune system. During the first three funding years of this new grant we have made substantial progress towards testing this hypothesis by: 1)Proving that specific bacterial products can inhibit lymphocyte function. 2) Identifying novel bacterial chromosomal regions in EPEC responsible for expression of these factor(s). 3) Identifying similar genes and inhibitory function in other enteric bacteria. 4) Delineating the characteristics and mechanisms of inhibition of lymphoid function by bacterial factors. 5) Developing the necessary tools to directly test this hypothesis in whole animal mode systems. The specific aims of this proposal will further test different aspects of our general hypothesis: The first aim win be provide direct evidence for the novel concept that enteric immunosuppressive bacterial factors can contribute to inflammatory bowl diseases by demonstrating the immunoregulatory effects of bacterial products in two different murine models in vivo. Aim 2. We will identity and express potential novel immunosuppressive bacterial products by expressing genes and surrounding genes identified by the transposon mutants that we have made that lack inhibitory activity. The precise identification of specific bacterial factors will allow for more detailed studies of immune mechanisms and bacterial pathogenesis. Aim 3. Using crude and specific bacterial products, we will further delineate the mechanism of action of these factors by examining cell cycle arrest, apoptosis, and regulation of lymphokine gene expression and effects on oral tolerance. Aim 4. Concurrently we will directly test the hypothesis that bacterial immunosuppressive factors play a role in human IBD by isolating bacteria from stool of patients and controls and testing by cross hybridization screening and functional testing of clinical isolates for inhibitory activity. As we demonstrate in the extensive progress report and preliminary new data in this proposal, we have assembled the necessary tools and a multidisciplinary team to accomplish these ambitious goals that further substantiate our novel hypothesis for the pathogenesis of IBD. Significance: The results of these studies will provide not only insights into the pathogenesis of immune and inflammatory diseases of the G1 tract, but will provide further understanding of basic of microbial pathogenesis in the gastrointestinal tract and possible new specific targets for therapy of IBD.